Lipolytic enzyme variant

ABSTRACT

Novel lipolytic enzymes are disclosed which are capable of removing substantial amounts of lard from a lard stained swatch in a one cycle wash. Preferred lipolytic enzymes are variants of the  Humicola lanuginosa  lipase which may be prepared by recombinant DNA techniques. The enzymes are advantageously used in detergent compositions.

FIELD OF THE INVENTION

[0001] The present invention relates to variants of fungal lipolytic enzymes, particularly variants with improved thermostability, and to methods of producing and using such variants.

BACKGROUND OF THE INVENTION

[0002] It is known to use fungal lipolytic enzymes, e.g. the lipase from Thermomyces lanuginosus (synonym Humicola lanuginosa), for various industrial purposes, e.g. to improve the efficiency of detergents and to eliminate pitch problems in pulp and paper production. In some situations, a lipolytic enzyme with improved thermostability is desirable (EP 374700, WO 9213130).

[0003] WO 92/05249, WO 92/19726 and WO 97/07202 disclose variants of the T. lanuginosus (H. lanuginosa) lipase.

SUMMARY OF THE INVENTION

[0004] The inventors have found that the thermostability of a fungal lipolytic enzyme can be improved by certain specified substitutions in the amino acid sequence.

[0005] Accordingly, the invention provides a variant of a parent fungal lipolytic enzyme, which variant comprises substitution of one or more specified amino acid residues and is more thermostable than the parent lipolytic enzyme. The invention also provides a method of producing a lipolytic enzyme variant comprising:

[0006] a) selecting a parent fungal lipolytic enzyme,

[0007] b) in the parent lipolytic enzyme substituting at least one specified amino acid residue,

[0008] c) optionally, substituting one or more amino acids other than b),

[0009] d) preparing the variant resulting from steps a)-c),

[0010] e) testing the thermostability of the variant,

[0011] f) selecting a variant having an increased thermostability, and

[0012] g) producing the selected variant.

[0013] The specified amino acid residues comprise amino acid residues corresponding to any of 21, 27, 29, 32, 34-42, 51, 54, 76, 84, 90-97, 101, 105, 111, 118, 125, 131, 135, 137, 162, 187, 189, 206-212, 216, 224-234, 242-252 and 256 of SEQ ID NO: 1.

[0014] The thermostability may particularly be increased by more than 4° C. The substitutions may be with a different amino acid residue, particularly one different from Pro.

DETAILED DESCRIPTION OF THE INVENTION

[0015] Parent Lipolytic Enzyme

[0016] The lipolytic enzyme to be used in the present invention is classified in EC 3.1.1 Carboxylic Ester Hydrolases according to Enzyme Nomenclature (available at http://www.chem.qmw.ac.uk/iubmb/enzyme). The substrate specificity may include activities such as EC 3.1.1.3 triacylglycerol lipase, EC 3.1.1.4 phospholipase A2, EC 3.1.1.5 lysophospholipase, EC 3.1.1.26 galactolipase, EC 3.1.1.32 phospholipase A1. EC 3.1.1.73 feruloyl esterase.

[0017] The parent lipolytic enzyme is fungal and has an amino acid sequence that can be aligned with SEQ ID NO: 1 which is the amino acid sequence shown in positions 1-269 of SEQ ID NO: 2 of U.S. Pat. No. 5,869,438 for the lipase from Thermomyces lanuginosus (synonym Humicola lanuginosa), described in EP 258 068 and EP 305 216. The parent lipolytic enzyme may particularly have an amino acid sequence with at least 50% homology with SEQ ID NO: 1. In addition to the lipase from T. lanuginosus, other examples are a lipase from Penicillium camembertii (P25234), lipase/phospholipase from Fusarium oxysporum (EP 130064, WO 98/26057), lipase from F. heterosporum (R87979), lysophospholipase from Aspergillus foetidus (W33009), phospholipase A1 from A. oryzae (JP-A 10-155493), lipase from A. oryzae (D85895), lipase/ferulic acid esterase from A. niger (Y09330), lipase/ferulic acid esterase from A. tubingensis (Y09331), lipase from A. tubingensis (WO 98/45453), lysophospholipase from A. niger (WO 98/31790), lipase from F. solanii having an isoelectric point of 6.9 and an apparent molecular weight of 30 kDa (WO 96/18729).

[0018] Other examples are the Zygomycetes family of lipases comprising lipases having at least 50% homology with the lipase of Rhizomucor miehei (P19515) having the sequence shown in SEQ ID NO: 2. This family also includes the lipases from Absidia reflexa, A. sporophora, A. corymbifera, A. blakesleeana, A. griseola (all described in WO 96/13578 and WO 97/27276) and Rhizopus oryzae (P21811). Numbers in parentheses indicate publication or accession to the EMBL, GenBank, GeneSeqp or Swiss-Prot databases.

[0019] Amino Acid Substitutions

[0020] The lipolytic enzyme variant of the invention comprises one or more substitutions of an amino acid residue in any of the regions described above. The substitution may, e.g., be made in any of the regions corresponding to 206-208, 224-228, 227-228, 227-231, 242-243 and 245-252 of SEQ ID NO: 1. The amino acid residue to be substituted may correspond to residue Y21, D27, P29, T32, A40, F51, S54, I76, R84, I90, G91, N94, N101, S105, D111, R118, R125, A131, H135, D137, N162, V187, T189, E210, G212, S216, G225, L227, I238 or P256 of SEQ ID NO: 1. Some particular substitutions of interest are those corresponding to D27N/R/S, P29S, T32S, F51I/L, I76V, R84C, I90L/V, G91A/N/S/T/W, L93F, N94K/R/S, F95I, D96G/N, N101D, D111A/G, R118M, A131V, H135Y, D137N, N162R, V187I, F211Y, S216P, S224I/Y, G225P, T226N, L227F/P/G/V, L227X, V228C/I, 238V and P256T of SEQ ID NO: 1.

[0021] The total number of substitutions in the above regions is typically not more than 10, e.g. one, two, three, four, five, six, seven or eight of said substitutions. In addition, the lipolytic enzyme variant of the invention may optionally include other modifications of the parent enzyme, typically not more than 10, e.g. not more than 5 such modifications. The variant may particularly have a total of not more than 10 amino acid modifications (particularly substitutions) compared to the parent lipolytic enzyme. The variant generally has a homology with the parent lipolytic enzyme of at least 80%, e.g. at least 85%, typically at least 90% or at least 95%.

[0022] Lipolytic Enzyme Variant

[0023] The variant has lipolytic enzyme activity, i.e. it is capable of hydrolyzing carboxylic ester bonds to release carboxylate (EC 3.1.1). It may particularly have lipase activity (triacylglycerol lipase activity, EC 3.1.1.3), i.e. hydrolytic activity for carboxylic ester bonds in triglycerides, e.g. 1,3-specific activity.

[0024] Specific Variants

[0025] The following are some examples of variants of the T. lanuginosus lipase. Corresponding substitutions may be made by making corresponding amino acid substitutions in other fungal lipolytic enzymes: D27N D111G + S216P L227F L227F + V228I G225P S224I + G225W + T226N + L227P + V228C S224Y + G225W + T226N + L227P + V228C D27R + D111G + S216P D27S + D111G + S216P D27N + D111A D27R + D111G + S216P + L227P + P256T D27R + D111G + S216P + L227G + P256T D27R + D111G + S216P + L227F + P256T D27R + D111G + S216P + L227V + P256T D27R + D111G + S216P + L227G D27R + D111G + S216P + L227X 027P + D111G + S216P + L227X

[0026] Thermostability

[0027] The thermostability can be measured at a relevant pH for the intended application using a suitable buffer. Examples of buffers and pH are: pH 10.0 (50 mM glycine buffer), pH 7.0 (50 mM HEPES Buffer) or pH 5.0 (50 mM sodium acetate as buffer).

[0028] For comparison, measurements should be made in the same buffer, at the same conditions and at the same protein concentration. Various methods can be used for measuring the thermostability:

[0029] Differential Scanning Calorimetry (DSC)

[0030] In DSC, the heating rate may be 90 degrees per hour. The sample may be purified to homogeneity, and the melting temperature (T_(M)) may be taken as an expression of the thermostability.

[0031] Residual Enzyme Activity

[0032] Alternatively, the thermostability can be determined by measuring residual lipolytic enzyme activity after incubation at selected temperatures. p-nitrophenyl ester in 10 mM Tris-HCl, pH 7.5 may be used as the substrate, as described in Giver et al., Proc. Natl. Acad. Sci. USA 95(1998)12809-12813 and Moore et al. Nat. Biotech. 14(1996) 458-467. Samples may be added periodically, or only one sample may be used with or without different additives to prevent or enhance denaturing, e.g. in a 96 well format.

[0033] CD Spectroscopy

[0034] CD spectroscopy as described e.g. in Yamaguchi et al. Protein engineering 9(1996)789-795. Typical enzyme concentration is around 1 mg/ml, Temperature between 5-80 degrees

[0035] Use of Variant

[0036] The lipolytic enzyme variants may be used in various processes, and some particular uses are described below. The variant is typically used at 60-95° C. (particularly 75-90° C., 70-90° C. or 70-85° C.) and pH 4.5-11 (particularly 4.5-8 or 5-6.5).

[0037] Use in the Paper and Pulp Industry

[0038] The lipase may be used in a process for avoiding pitch troubles in a process for the production of mechanical pulp or a paper-making process using mechanical pulp, which comprises adding the lipase to the pulp and incubating. The lipase addition may take place in the so-called white water (recycled process water). It may also be used to remove ink from used paper. The improved thermostability allows the variant to be used at a higher temperature, generally preferred in the industry. This may be done in analogy with WO 9213130, WO 9207138, JP 2160984 A, EP 374700.

[0039] Use in Cereal-Based Food Products

[0040] The lipolytic enzyme variant may be added to a dough, and the dough may be used to prepare a baked product (particularly bread), pasta or noodles. The improved thermostability of the variant allows it to remain active for a longer time during the heating step (baking, boiling or frying). This may be done in analogy with WO 94/04035, WO 00/32758 , PCT/DK 01/00472, EP 1057415.

[0041] The addition of the variant may lead to improved dough stabilization, i.e. a larger loaf volume of the baked product and/or a better shape retention during baking, particularly in a stressed system, e.g. in the case of over-proofing or over-mixing. It may also lead to a lower initial firmness and/or a more uniform and fine crumb, improved crumb structure (finer crumb, thinner cell walls, more rounded cells), of the baked product, and it may further improve dough properties, e.g. a less soft dough, higher elasticity, lower extensibility.

[0042] Use in the Fat and Oil Industry

[0043] The lipolytic enzyme variant may be used as a catalyst in organic synthesis, e.g. in a process for hydrolyzing, synthesizing or interesterifying an ester, comprising reacting the ester with water, reacting an acid with an alcohol or interesterifying the ester with an acid, an alcohol or a second ester in the presence of the lipolytic enzyme variant. Favorably, the improved thermostability allows the process to be conducted at a relatively high temperature which may be favorable to increase the rate of reaction and to process high-melting substrates.

[0044] The ester may be a carboxylic acid ester, e.g. a triglyceride. The interesterification may be done in the presence or absence of a solvent. The enzyme may be used in immobilized form. The process may be conducted in analogy with WO 8802775, U.S. Pat. No. 6,156,548, U.S. Pat. No. 5,776,741, EP 792106, EP 93602, or EP 307154.

[0045] Use in Textile Industry

[0046] The variant may be used in a process for enzymatic removal of hydrophobic esters from fabrics, which process comprises treating the fabric with an amount of the lipolytic enzyme effective to achieve removal of hydrophobic esters from fabric. The treatment may be done at a temperature of 75° C. or above, e.g. for a period of 1-24 hours. The treatment may be preceded by impregnating the fabric with an aqueous solution of the lipase variant to a liquor pick-up ratio of 50-200%, and may be followed by washing and rinsing to remove the fatty acids.

[0047] The process may be conducted in analogy with U.S. Pat. No. 5,578,489 or U.S. Pat. No. 6,077,316.

[0048] Use in Detergents

[0049] The variant may be used as a detergent additive, e.g. at a concentration (expressed as pure enzyme protein) of 0.001-10 (e.g. 0.01-1) mg per gram of detergent or 0.001-100 (e.g. 0.01-10) mg per liter of wash liquor. This may be done in analogy with WO 97/04079, WO 97/07202, WO 97/41212, WO 98/08939 and WO 97/43375.

[0050] Use for Leather

[0051] The variants of the invention can also be used in the leather industry in analogy with GB 2233665 or EP 505920.

[0052] Nomenclature for Amino Acid Substitutions

[0053] The nomenclature used herein for defining amino acid substitutions uses the single-letter code, as described in WO 92/05249.

[0054] Thus, D27N indicates substitution of D in position 27 with N. D27N/R indicates a substitution of D27 with N or R. L227X indicates a substitution of L227 with any other amino acid. D27N+D111A indicates a combination of the two substitutions.

[0055] Homology and Alignment

[0056] For purposes of the present invention, the degree of homology may be suitably determined by means of computer programs known in the art, such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-45), using GAP with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.

[0057] In the present invention, corresponding (or homologous) positions in the lipase sequences of Rhizomucor miehei (rhimi), Rhizopus delemar (rhidl), Thermomyces lanuginosa (former; Humicola lanuginosa) (SP400), Penicillium camembertii (Pcl) and Fusarium oxysporum (FoLnp11), are defined by the alignment shown in FIG. 1 of WO 00/32758.

[0058] To find the homologous positions in lipase sequences not shown in the alignment, the sequence of interest is aligned to the sequences shown in FIG. 1. The new sequence is aligned to the present alignment in FIG. 1 by using the GAP alignment to the most homologous sequence found by the GAP program. GAP is provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-45). The following settings are used for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.

[0059] Procedure for Obtaining Thermostable Variants

[0060] Variants of a lipolytic enzyme can be obtained by methods known in the art, such as site-directed mutagenesis, random mutagenesis or localized mutagenesis, e.g. as described in WO 9522615 or WO 0032758.

[0061] Thermostable variants of a given parent lipolytic enzyme can be obtained by the following standard procedure:

[0062] Mutagenesis (error-prone, doped oligo, spiked oligo)

[0063] Primary Screening

[0064] Identification of more temperature stable mutants

[0065] Maintenance (glycerol culture, LB-Amp plates, Mini-Prep)

[0066] Streaking out on another assay plate—secondary screening (1 degree higher then primary screening)

[0067] DNA Sequencing

[0068] Transformation in Aspergillus

[0069] Cultivation in 100 ml scale, purification, DSC

[0070] Primary Screening Assay

[0071] The following assay method is used to screen lipolytic enzyme variants and identify variants with improved thermostability.

[0072]E. coli cells harboring variants of a lipolytic enzyme gene are prepared, e.g. by error-prone PCR, random mutagenesis or localized random mutagenesis or by a combination of beneficial mutants and saturation mutagenesis.

[0073] The assay is performed with filters on top of a LB agar plate. E. coli cells are grown on cellulose acetate filters supplied with nutrients from the LB agar plate and under the selection pressure of ampicillin supplied with the LB agar. Proteins including the desired enzyme are collected on a nitrocellulose filter between LB agar and cellulose acetate filter. This nitrocellulose filter is incubated in a buffer of desired pH (generally 6.0) and at the desired temperature for 15 minutes (e. g. 78 degrees for the T. lanuginosus lipase). After quenching the filters in ice-water, the residual lipase activity is determined through the cleavage of indole acetate and the subsequent coloration of the reaction product with nitro-blue tetrazolium chloride as described by Kynclova, E et al. (Journal of Molecular Recognition 8 (1995)139-145).

[0074] The heat treatment applied is adjusted so that the parent generation is slightly active, approximately 5-10% compared to samples incubated at room temperature. This facilitates the identification of beneficial mutants.

EXAMPLES Example 1 Expression of Lipase

[0075] Plasmid pMT2188

[0076] The Aspergillus oryzae expression plasmid pCaHj 483 (WO 98/00529) consists of an expression cassette based on the Aspergillus niger neutral amylase II promoter fused to the Aspergillus nidulans triose phosphate isomerase non translated leader sequence (Pna2/tpi) and the A. niger amyloglycosidase terminater (Tamg). Also present on the plasmid is the Aspergillus selective marker amdS from A. nidulans enabling growth on acetamide as sole nitrogen source. These elements are cloned into the E. coli vector pUC19 (New England Biolabs). The ampicillin resistance marker enabling selection in E. coli of this plasmid was replaced with the URA3 marker of Saccharomyces cerevisiae that can complement a pyrF mutation in E. coli, the replacement was done in the following way:

[0077] The pUC19 origin of replication was PCR amplified from pCaHj483 with the primers 142779 (SEQ ID NO: 3) and 142780 (SEQ ID NO: 4).

[0078] Primer 142780 introduces a BbuI site in the PCR fragment. The Expand PCR system (Roche Molecular Biochemicals, Basel, Switserland) was used for the amplification following the manufacturers instructions for this and the subsequent PCR amplifications.

[0079] The URA3 gene was amplified from the general S. cerevisiae cloning vector pYES2 (Invitrogen corporation, Carlsbad, Calif., USA) using the primers 140288 (SEQ ID 5) and 142778 (SEQ ID 6).

[0080] Primer 140288 introduces an EcoRI site in the PCR fragment. The two PCR fragments were fused by mixing them and amplifying using the primers 142780 and 140288 in the splicing by overlap method (Horton et al (1989) Gene, 77, 61-68).

[0081] The resulting fragment was digested with EcoRI and BbuI and ligated to the largest fragment of pCaHj 483 digested with the same enzymes. The ligation mixture was used to transform the pyrF E. coli strain DB6507 (ATCC 35673) made competent by the method of Mandel and Higa (Mandel, M. and A. Higa (1970) J. Mol. Biol. 45, 154). Transformants were selected on solid M9 medium (Sambrook et. al (1989) Molecular cloning, a laboratory manual, 2. edition, Cold Spring Harbor Laboratory Press) supplemented with 1 g/l casaminoacids, 500 μg/l thiamine and 10 mg/l kanamycin.

[0082] A plasmid from a selected transformant was termed pCaHj 527. ThePna2/tpi promoter present on pCaHj527 was subjected to site directed mutagenises by a simple PCR approach.

[0083] Nucleotide 134-144 was altered from SEQ ID NO: 7 to SEQ ID NO: 8 using the mutagenic primer 141223 (SEQ ID NO: 9).

[0084] Nucleotide 423-436 was altered from SEQ ID NO: 10 to SEQ ID NO: 11 using the mutagenic primer 141222 (SEQ ID 12).

[0085] The resulting plasmid was termed pMT2188.

[0086] Plasmid pENI1849

[0087] Plasmid pENI1849 was made in order to truncate the pyrG gene to the essential sequences for pyrG expression, in order to decrease the size of the plasmid, thus improving transformation frequency. A PCR fragment (app. 1800 bp) was made using pENI1299 (described in WO 00/24883) as template and the primers 270999J8 (SEQ ID 13) and 270999J9 (SEQ ID 14).

[0088] The PCR-fragment was cut with the restriction enzymes StuI and SphI, and cloned into pENI1298 (described in WO 0024883), also cut with StuI and SphI; the cloning was verified by sequencing.

[0089] Plasmid pENI1861

[0090] Plasmid pENI1861 was made in order to have the state of the art Aspergillus promoter in the expression plasmid, as well as a number of unique restriction sites for cloning.

[0091] A PCR fragment (app. 620 bp) was made using pMT2188 (see above) as template and the primers 051199J1 (SEQ ID 15) and 1298TAKA (SEQ ID 16).

[0092] The fragment was cut BssHII and Bgl II, and cloned into pEN11849 which was also cut with BssHII and Bgl II. The cloning was verified by sequencing.

[0093] Plasmid pENI1902

[0094] Plasmid pENI1902 was made in order to have a promoter that works in both E. coli and Aspergillus. This was done by unique site elimination using the “Chameleon double stranded site-directed mutagenesis kit” as recommended by Stratagene®.

[0095] Plasmid pENI1861 was used as template and the following primers with 5′ phosphorylation were used as selection primers: 177996 (SEQ ID 17), 135640 (SEQ ID 18) and 135638 (SEQ ID 19).

[0096] The 080399J19 primer (SEQ ID NO: 20) with 5′ phosphorylation was used as mutagenic primer to introduce a −35 and −10 promoter consensus sequence (from E. coli) in the Aspergillus expression promoter. Introduction of the mutations was verified by sequencing.

[0097] Plasmid pSMin001

[0098] Plasmid pSMin001 was made in order to permit the expression of the T. lanuginosus lipase in E. coli and Aspergillus.

[0099] Plasmid pAHL (described in WO 9205249) was used as template for PCR to amplify the T. lanuginosus lipase gene with the following Primers: 19671 (SEQ ID NO: 21) and 991213J5 (SEQ ID NO: 22). Primer 991213J5 introduced a SacII site into the PCR fragment. The PCR fragment (appr. 1100 bp) was cut with BamHI and SacII and cloned into pEni1902 cut with the same enzymes. The cloning was verified by DNA sequencing. The plasmid was transformed in E. coli DH5α, and lipase expression was detected by using the described filter assay.

[0100] Using this newly developed plasmid it was possible to express the desired enzyme in Aspergillus without any modification. The achieved expression rates in E. coli were quite low, but sufficient for the screening assay.

Example 2 Production of Thermostable Lipase Variants

[0101] Several techniques were used to create diversity in the T. lanuginosus lipase gene: error-prone PCR, localized random mutagenesis with the aid of doped oligonucleotides, and site-directed mutagenesis.

[0102] Variants exhibiting higher temperature stability were selected by the primary assay described above, and were cultivated in LB media and streaked out again on assay plates as described above for a secondary screening. The assay in the secondary screening was performed with a 1-1.5 degrees higher temperature. The DNA of mutants still active under these conditions were sequenced and transformed into Aspergillus to obtain a higher amount of protein, followed by a chromatographic purification. The purified enzyme was used for DSC analysis to prove the enhancement of the stability.

[0103] Next, amino acid substitutions found in the beneficial variants were combined, and saturation mutagenesis was used to ensure that all 20 amino acids were introduced in the desired positions.

Example 3 Thermostability of Lipase Variants

[0104] All samples identified as more thermostable in the primary and secondary screening In Example 2 were purified to homogeneity, and their stability was checked by differential scanning calorimetry (DSC) at pH 5.0 and/or 7.0 to determine the stability of the protein, given by its melting temperature (T_(M)). The parent lipase from T. lanuginosus was included for comparison.

[0105] Eight variants were found to have increased thermostability at pH 5.0, four variants showing an increase of more than 4° C. Two variants were tested at pH 7.0 and found to have improved thermostability.

Example 4 Thermostability of Lipase Variants by DSC

[0106] A number of variants of the T. lanuginosus lipase were prepared and purified, and the thermostability was checked by differential scanning calorimetry (DSC) at pH 5.0 to determine the stability of the protein, given by its melting temperature (T_(M)). The parent lipase from T lanuginosus was included for comparison.

[0107] The following variants were found to be more thermostable than the parent lipase: D111G + S216P D27N L227F S224I + G225W + T226N + L227P + V228C L227F + V228I G225P W221C + G246C

[0108] The following variants were found to be more thermostable than the parent lipase with at least 4° C. increase of the melting temperature. D27R + D111G + S216P D27N + D111A D27R + D111G + S216P + L227G + P256T D27R + D111G + S216P + L227F + P256T D27R + D111G + S216P + L227G D27S + D111G + S216P D27R + D111A + S216P + L227G + P256T D27R + D111G + S216P + G225P + L227G + P256T D27R + T37S + D111G + S216P + L227G + P256T D27R + N39F + D111G + S216P + L227G + P256T D27R + G38C + D111G + S216P + L227G + P256T D27R + D111G + S216P + L227G + T2441 + P256T D27R + G91A + D111G + S216P + L227G + P256T N25I + D27R + D111A + S216P + L227G + P256T N25L + D27R + D111A + S216P + L227G + P256T N26D + D27R + D111A + S216P + L227G + P256T D27R + K46R + D111A + S216P + L227G + P256T D27R + V60N + D111A + S216P + L227G + P256T D27R + D111A + P136A + S216P + L227G + P256T D27R + D111A + S216P + L227G + P256T + I265F D27R + S58Y + D111A + S216P + L227G + P256T + N26D + D27R + E56Q + D111A + S216P + L227G + P256T D27R + G91A + D96E + L97Q + D111A + S216P + L227G + P256T D27R + G91A + D111A + S216P + L227G + P256T + D27R + G91T + N94S + D111A + S216P + L227G + P256T D27R + G91S + D111A + S216P + L227G + P256T + D27R + G91N + D111A + S216P + L227G + P256T D27R + D96E + D111A + S216P + L227G + P256T D27R + I90L + G91A + N94K + D111A + S216P + L227G + P256T D27R + G91S + F95V + D111A + S216P + L227G + P256T

Example 5 Thermostability by Plate Assay

[0109] A number of variants of the T. lanuginosus lipase were prepared and tested for thermostability as described above under “primary screening assay”. The parent lipase from T. lanuginosus was included for comparison.

[0110] The following variants were found to be more thermostable than the parent lipase: D27R + I90V + G91S + D111A + S216P + L227G + P256T D27R + G91N + N94R + D111A + S216P + L227G + P256T D27R + I90L + L93F + 096N + D111A + S216P + L227G + P256T D27R + I90L + G91A + D96E + D111A + S216P + L227G + P256T D27R + G91S + L93F + D111A + S216P + L227G + P256T D27R + G91T + N94K + D111A + S216P + L227G + P256T D27R + G91T + 0111A + S216P + L227G + P256T D27R + L93F + D111A + D137N + S216P + L227G + P256T D27R + G91S + 096N + D111A + S216P + L227G + P256T D27R + G91W + D111A + S216P + L227G + P256T D27R + I90L + G91T + D111A + S216P + L227G + P256T D27R + G91S + L93F + N94R + D96G + D111A + S216P + L227G + P256T D27R + G91T + D96N + D111A + S216P + L227G + P256T D27R + I90V + G91T + L93F + N94K + D111A + S216P + L227G + P256T D27R + L93V + D111A + S216P + L227G + P256T D27R + G91S + N94K + D111A + S216P + L227G + P256T D27R + I90L + G91T + D111A + S216P + L227G + P256T D27R + G91S + L93F + F951 + D96N + D111A + S216P + L227G + P256T D27R + D111A + V187I + S216P + L227G + P256T D27R + D111A + F211Y + S216P + L227G + P256T D27R + R118M + D111A + A131V + S216P + L227G + P256T D27R + P29S + R84C + D111A + H135Y + S216P + L227G + P256T D27R + T32S + D111A + H135Y + S216P + L227G + P256T D27R + G91R + D111A + 1238V + S216P + L227G + P256T D27R + F51I + I76V + N101D + D111A + N162R + S216P + L227G + P256T D27R + F51L + D111A + S216P + L227G + P256T

[0111]

1 22 1 269 PRT Thermomyces lanuginosus 1 Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr 1 5 10 15 Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala Gly Thr 20 25 30 Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu Val Glu Lys Ala Asp 35 40 45 Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr 50 55 60 Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe 65 70 75 80 Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn Phe Asp 85 90 95 Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Asp Gly 100 105 110 Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val 115 120 125 Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly 130 135 140 His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg 145 150 155 160 Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val 165 170 175 Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr 180 185 190 Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro 195 200 205 Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile Lys Ser 210 215 220 Gly Thr Leu Val Pro Val Thr Arg Asn Asp Ile Val Lys Ile Glu Gly 225 230 235 240 Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Asp Ile Pro 245 250 255 Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr Cys Leu 260 265 2 269 PRT Rhizomucor miehei 2 Ser Ile Asp Gly Gly Ile Arg Ala Ala Thr Ser Gln Glu Ile Asn Glu 1 5 10 15 Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val 20 25 30 Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp 35 40 45 Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala 50 55 60 Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg 65 70 75 80 Gly Ser Ser Ser Ile Arg Asn Ala Ile Ala Asp Leu Thr Phe Val Pro 85 90 95 Val Ser Tyr Pro Pro Val Ser Gly Thr Lys Val His Lys Gly Phe Leu 100 105 110 Asp Ser Tyr Gly Glu Val Gln Asn Glu Leu Val Ala Thr Val Leu Asp 115 120 125 Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser 130 135 140 Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg 145 150 155 160 Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln 165 170 175 Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly 180 185 190 Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu 195 200 205 Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile 210 215 220 Thr Asp Asn Ser Pro Glu Thr Val Gln Val Cys Thr Ser Asp Leu Glu 225 230 235 240 Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp 245 250 255 His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Ser 260 265 3 31 DNA Artificial Sequence Primer 3 ttgaattgaa aatagattga tttaaaactt c 31 4 25 DNA Artificial Sequence Primer 4 ttgcatgcgt aatcatggtc atagc 25 5 26 DNA Artificial Sequence Primer 5 ttgaattcat gggtaataac tgatat 26 6 32 DNA Artificial Sequence Primer 6 aaatcaatct attttcaatt caattcatca tt 32 7 11 DNA Artificial Sequence Primer 7 gtactaaaac c 11 8 11 DNA Artificial Sequence Primer 8 ccgttaaatt t 11 9 45 DNA Artificial Sequence Primer 9 ggatgctgtt gactccggaa atttaacggt ttggtcttgc atccc 45 10 14 DNA Artificial Sequence Primer 10 atgcaattta aact 14 11 14 DNA Artificial Sequence Primer 11 cggcaattta acgg 14 12 44 DNA Artificial Sequence Primer 12 ggtattgtcc tgcagacggc aatttaacgg cttctgcgaa tcgc 44 13 26 DNA Artificial Sequence Primer 13 tctgtgaggc ctatggatct cagaac 26 14 27 DNA Artificial Sequence Primer 14 gatgctgcat gcacaactgc acctcag 27 15 59 DNA Artificial Sequence Primer 15 cctctagatc tcgagctcgg tcaccggtgg cctccgcggc cgctggatcc ccagttgtg 59 16 33 DNA Artificial Sequence Primer 16 gcaagcgcgc gcaatacatg gtgttttgat cat 33 17 30 DNA Artificial Sequence Primer 17 gaatgacttg gttgacgcgt caccagtcac 30 18 25 DNA Artificial Sequence Primer 18 cttattagta ggttggtact tcgag 25 19 37 DNA Artificial Sequence Primer 19 gtccccagag tagtgtcact atgtcgaggc agttaag 37 20 64 DNA Artificial Sequence Primer 20 gtatgtccct tgacaatgcg atgtatcaca tgatataatt actagcaagg gaagccgtgc 60 ttgg 64 21 24 DNA Artificial Sequence Primer 21 ctcccttctc tgaacaataa accc 24 22 66 DNA Artificial Sequence Primer 22 cctctagatc tcgagctcgg tcaccggtgg cctccgcggc cgctgcgcca ggtgtcagtc 60 accctc 66 

1. A variant of a parent fungal lipolytic enzyme, wherein the variant a) has an amino acid sequence which compared to the parent lipolytic enzyme comprises substitution of an amino acid residue corresponding to any of amino acids 21, 27, 29, 32, 34-42, 51, 54, 76, 84, 90-97, 101, 105, 111, 118, 125, 131, 135, 137, 162, 187, 189, 206-212, 216, 224-234, 242-252 and 256 of SEQ ID NO: 1, and b) is more thermostable than the parent lipolytic enzyme.
 2. The variant of the preceding claim which is at least 4° C. more thermostable than the parent lipolytic enzyme.
 3. The variant of either preceding claim, wherein the amino acid residue is substituted with an amino acid residue different from Pro.
 4. The variant of any preceding claim wherein the parent lipolytic enzyme has at least 50% homology with SEQ ID NO:
 1. 5. The variant of the preceding claim wherein the parent lipolytic enzyme is the lipase produced by Thermomyces lanuginosus DSM 4109 and having the amino acid sequence of SEQ ID NO:
 1. 6. The variant of either preceding claim which comprises substitution of an amino acid residue corresponding to Y21, D27, P29, T32, A40, F51, S54, I76, R84, I90, G91, N94, N101, S105, D111, R118, R125, A131, H135, D137, N162, V187, T189, E210, G212, S216, G225, L227, I238 or P256 of SEQ ID NO:1.
 7. The variant of any preceding claim which comprises one or more substitutions corresponding to D27N/R/S, P29S, T32S, F51I/L, I76V, R84C, I90L/N, G91A/N/S/T/W, L93F, N94K/R/S, F95I, D96G/N, N101D, D111A/G, R118M, A131V, H135Y, D137N, N162R, V187I, F211Y, S216P, S224I/Y, G225P, T226N, L227F/P/G/V, L227X, V228C/I, 238V and P256T of SEQ ID NO:
 1. 8. The variant of any preceding claim which has one, two, three, four, five, six, seven or eight of said substitutions.
 9. The variant of any preceding claim which further comprises one or more substitutions of amino acid residues other than those listed in claim 1, preferably 1-5 such substitutions.
 10. The variant of any preceding claim which comprises substitutions corresponding to the following in SEQ ID NO: 1: a) D27N b) D111G+S216P c) L227F d) L227F+V228I e) G225P f) S224I+G225W+T226N+L227P+V228C g) S224Y+G225W+T226N+L227P+V228C h) D27R+D111G+S216P i) D27S+D111G+S216P j) D27N+D111A k) D27R+D111G+S216P+L227P+P256T l) D27R+D111G+S216P+L227G+P256T m) D27R+D111G+S216P+L227F+P256T n) D27R+D111G+S216P+L227V+P256T o) D27R+D111G+S216P+L227G p) D27R+D111G+S216P+L227X q) D27P+D111G+S216P+L227X r) S224I+G225W+T226N+L227P+V228C s) W221C+G246C t) D27R+D111G+S216P u) D27N+D111A v) D27R+D111G+S216P+L227G+P256T w) D27R+D111G+S216P+L227F+P256T x) D27R+D111G+S216P+L227G y) D27S+D111G+S216P z) D27R+D111A+S216P+L227G+P256T aa) D27R+D111G+S216P+G225P+L227G+P256T bb) D27R+T37S+D111G+S216P+L227G+P256T cc) D27R+N39F+D111G+S216P+L227G+P256T dd) D27R+G38C+D111G+S216P+L227G+P256T ee) D27R+D111G+S216P+L227G+T2441+P256T ff) D27R+G91A+D111G+S216P+L227G+P256T gg) N25I+D27R+D111A+S216P+L227G+P256T hh) N25L+D27R+D111A+S216P+L227G+P256T ii) N26D+D27R+D111A+S216P+L227G+P256T jj) D27R+K46R+D111A+S216P+L227G+P256T kk) D27R+V60N+D111A+S216P+L227G+P256T ll) D27R+D111A+P136A+S216P+L227G+P256T mm) D27R+D111A+S216P+L227G+P256T+I265F nn) D27R+S58Y+D111A+S216P+L227G+P256T+ oo) N26D+D27R+E56Q+D111A+S216P+L227G+P256T pp) D27R+G91A+D96E+L97Q+D111A+S216P+L227G+P256T qq) D27R+G91A+D111A+S216P+L227G+P256T+ rr) D27R+G91T+N94S+D111A+S216P+L227G+P256T ss) D27R+G91S+D111A+S216P+L227G+P256T+ tt) D27R+G91N+D111A+S216P+L227G+P256T uu) D27R+D96E+D111A+S216P+L227G+P256T vv) D27R+I90L+G91A+N94K+D111A+S216P+L227G+P256T ww) D27R+G91S+F95V+D111A+S216P+L227G+P256T
 11. The variant of any preceding claim having a denaturation temperature which is at least 5° C. higher than the parent lipolytic enzyme, preferably measured at pH 5-7.
 12. A DNA sequence encoding the variant of any preceding claim.
 13. A vector comprising the DNA sequence of the preceding claim.
 14. A transformed host cell harboring the DNA sequence of claim 12 or the vector of claim
 13. 15. A method of producing the variant of any of claims 1-11 comprising a) cultivating the cell of claim 14 so as to express and preferably secrete the variant, and b) recovering the variant.
 16. A method of producing a lipolytic enzyme variant comprising: a) selecting a parent fungal lipolytic enzyme, b) in the parent lipolytic enzyme substituting at least one amino acid residue corresponding to any of 21, 27, 29, 32, 34-42, 51, 54, 76, 84, 90-97, 101, 105, 111, 118, 125, 131, 135, 137, 162, 187, 189, 206-212, 216, 224-234, 242-252 and 256 of SEQ ID NO: 1, c) optionally, substituting one or more amino acids other than b), d) preparing the variant resulting from steps a)-c), e) testing the thermostability of the variant, f) selecting a variant having an increased thermostability, and g) producing the selected variant.
 17. The method of the preceding claim wherein the parent lipolytic enzyme has at least 50% homology with SEQ ID NO:
 1. 18. The method of the preceding claim wherein the parent lipolytic enzyme is the lipase produced by Thermomyces lanuginosus DSM 4109 and having the amino acid sequence of SEQ ID NO:
 1. 19. The method of either preceding claim which comprises substituting an amino acid residue corresponding to Y21, D27, P29, T32, A40, F51, S54, I76, R84, I90, G91, N94, N101, S105, D111, R118, R125, A131, H135, D137, N162, V187, T189, E210, G212, S216, G225, L227, I238 or P256 of SEQ ID NO:1.
 20. The method of the preceding claim which comprises substituting an amino acid residue corresponding to D27N/R/S, P29S, T32S, F51I/L, I76V, R84C, I90L/V, G91A/N/S/T/W, L93F, N94K/R/S, F95I, D96G/N, N101D, D111A/G, R118M, A131V, H135Y, D137N, N162R, V187I, F211Y, S216P, S224I/Y, G225P, T226N, L227F/P/G/V, L227X, V228C/I, 238V and P256T of SEQ ID NO:
 1. 21. A process for hydrolyzing a carboxylic acid ester, comprising incubating the ester with the lipase of any of claims 1-11 in the presence of water.
 22. A process for controlling pitch troubles in a process for the production of mechanical pulp or a paper-making process using mechanical pulp, which comprises adding the lipase of any of claims 1-11 to the pulp and incubating.
 23. The process of either preceding claim wherein the incubation is done at a temperature 60-95° C., particularly 75-90° C.
 24. The process of any preceding claim, wherein the incubation is done at a pH in the range 4.5-11, particularly 5-6.5.
 25. A process for preparing a dough or a baked product prepared from the dough, comprising adding the lipolytic enzyme of any of claims 1-11 to the dough.
 26. A process for hydrolyzing, synthesizing or interesterifying an ester, comprising reacting the ester with water, reacting an acid with an alcohol or interesterifying the ester with an acid, an alcohol or a second ester in the presence of the lipolytic enzyme of any of claims 1-11.
 27. A process for enzymatic removal of hydrophobic esters from fabrics, which process comprises treating the fabric with an amount of the lipolytic enzyme of any of claims 1-11 effective to achieve removal of hydrophobic esters from fabric. 